Insects cost farmers billions of dollars annually in crop losses and in the expense of keeping these pests under control. The losses caused by insect pests in agricultural production environments include decrease in crop yield, reduced crop quality, and increased harvesting costs.
Chemical pesticides have provided an effective method of pest control; however, the public has become concerned about the amount of residual chemicals which might be found in food, ground water, and the environment. Therefore, synthetic chemical pesticides are being increasingly scrutinized, and correctly so, for their potential toxic environmental consequences. Synthetic chemical pesticides can poison the soil and underlying aquifers, pollute surface waters as a result of runoff, and destroy non-target life forms. Synthetic chemical control agents have the further disadvantage of presenting public safety hazards when they are applied in areas where pets, farm animals, or children may come into contact with them. They may also provide health hazards to applicants, especially if the proper application techniques are not followed. Regulatory agencies around the world are restricting and/or banning the uses of many pesticides and particularly the organic synthetic chemical pesticides which are persistent in the environment and enter the food chain. Examples of widely used synthetic chemical pesticides include the organochlorines, e.g., DDT, mirex, kepone, lindane, aldrin, chlordane, aldicarb, and dieldrin; the organophosphates, e.g., chlorpyrifos, parathion, malathion, and diazinon; and carbamates. Stringent new restrictions on the use of pesticides and the elimination of some effective pesticides from the market place could limit economical and effective options for controlling costly pests.
Because of the problems associated with the use of organic synthetic chemical pesticides, there exists a clear need to limit the use of these agents and a need to identify alternative control agents. The replacement of synthetic chemical pesticides, or combination of these agents with biological pesticides, could reduce the levels of toxic chemicals in the environment. A biological pesticidal agent that is enjoying increasing popularity is the soil microbe Bacillus thuringiensis (B.t.). Bacillus thuringiensis is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and specific in their toxic activity. Certain B.t. toxin genes have been isolated and sequenced, and recombinant DNA-based B.t. products have been produced and approved for use. In addition, with the use of genetic engineering techniques, new approaches for delivering these B.t. endotoxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for pest resistance and the use of stabilized intact microbial cells as B.t. endotoxin delivery vehicles. Until the last ten years, commercial use of B.t. pesticides has been largely restricted to a narrow range of lepidopteran (caterpillar) pests. In recent years, however, investigators have discovered B.t. pesticides with specificities for a much broader range of pests.
Unfortunately, certain insects are refractory to the effects of B.t. and/or insects may develop resistance to B.t. The former includes insects such as boll weevil and black cutworm as well as adult insects of most species which heretofore have demonstrated no apparent significant sensitivity to B.t. .delta.-endotoxins. With respect to the latter, resistance management strategies in B.t. transgene plant technology have ascended to a prominent position. There remains, however, a great need to identify new insect control methods which are effective and also safe for use in the environment.
One possible approach to insect control involves the disruption of vital metabolic functions of the insect. Steroid compounds play an important role in the growth and development of insects. Insects are unable to form the cyclopentanoperhydrophenanthrene ring structure of steroids. As such they are dependent on dietary sources of steroids (cholesterol and/or .beta.-sitosterol) for subsequent elaboration of developmental steroids including ecdysone and 20-hydroxyecdysone. Ecdysone and 20-hydroxyecdysone are pivotal hormones in insect metamorphosis. Ecdysone oxidase mediates the oxidation of ecdysone and 20-hydroxyecdysone to 3-dehydoxyecdysone and 3-dehydro-20-hydroxyecdysone, respectively, plus H.sub.2 O.sub.2. Insects appear to be unique in this oxidation reaction. The reaction products have marginal molting activity and no other known hormonal activity, thus ecdysone oxidase is believed to participate in inactivation pathways of steroid catabolism. Ecdysone oxidase is localized in the fat body and cytosol of the gut of insects. Studies have shown that exogenously administered ecdysone and 20-hydroxyecdysone can have a profound effect on insect development and may even result in death (Tanaka, 1993).
The gene encoding cholesterol exidase has been cloned into plants (Purcell, 1994; Corbin, 1994). However, mammals are dependent on cholesterol as precursor for the elaboration of steroid hormones (corticosterone, sex hormones, etc.). Such presentation of an active enzyme in planta may present safety issues because of the potential for interference with mammalian steroid elaboration.